Direct and efficient production of ethanol from cellulosic material with a yeast strain displaying cellulolytic enzymes

Abstract

For direct and efficient ethanol production from cellulosic materials, we constructed a novel cellulose-degrading yeast strain by genetically codisplaying two cellulolytic enzymes on the cell surface of Saccharomyces cerevisiae. By using a cell surface engineering system based on alpha-agglutinin, endoglucanase II (EGII) from the filamentous fungus Trichoderma reesei QM9414 was displayed on the cell surface as a fusion protein containing an RGSHis6 (Arg-Gly-Ser-His(6)) peptide tag in the N-terminal region. EGII activity was detected in the cell pellet fraction but not in the culture supernatant. Localization of the RGSHis6-EGII-alpha-agglutinin fusion protein on the cell surface was confirmed by immunofluorescence microscopy. The yeast strain displaying EGII showed significantly elevated hydrolytic activity toward barley beta-glucan, a linear polysaccharide composed of an average of 1,200 glucose residues. In a further step, EGII and beta-glucosidase 1 from Aspergillus aculeatus No. F-50 were codisplayed on the cell surface. The resulting yeast cells could grow in synthetic medium containing beta-glucan as the sole carbon source and could directly ferment 45 g of beta-glucan per liter to produce 16.5 g of ethanol per liter within about 50 h. The yield in terms of grams of ethanol produced per gram of carbohydrate utilized was 0.48 g/g, which corresponds to 93.3% of the theoretical yield. This result indicates that efficient simultaneous saccharification and fermentation of cellulose to ethanol are carried out by a recombinant yeast cells displaying cellulolytic enzymes.

FIG. 1.

(A) Expression plasmid for display of T. reesei EGII (pEG23u31H6) on the yeast cell surface. (B) Immunofluorescence labeling of transformed cells: Nomarski differential interference micrographs (column 1) and immunofluorescence micrographs (columns 2 and 3) of S. cerevisiae MT8-1/pMUCS (control), MT8-1/pEG23u31H6, MT8-1/pBG211 and MT8-1/pBG211/pEG23u31H6. Cells were labeled with mouse anti-RGSHis4 antibody and goat anti-mouse IgG conjugated with Alexa Fluor 546 and with rabbit anti-A. aculeatus BGL1 antibody and goat anti-rabbit IgG conjugated with FITC.

FIG. 2.

HPLC analysis of hydrolysis products released from barley β-glucan. (A) MT8-1/pMUCS (control); (B) MT8-1/pEG23u31H6; (C) MT8-1/pBG211/pEG23u31H6. 1, glucose; 2, cellobiose; 3, cellotriose; 4, cellotetraose; 5, cellopentaose.

FIG. 3.

Time course of cell growth in synthetic medium containing barley β-glucan as the sole carbon source. Symbols: ♦, MT8-1/pHCS5/pMUCS (control); ▪, MT8-1/pHCS5/pEG23u31H6; ▴, MT8-1/pBG211/pMUCS; •, MT8-1/pBG211/pEG23u31H6. Cell growth was monitored by determining the OD₆₀₀ of the culture broth. The data points represent the averages of two independent experiments.

FIG. 4.

Time course of production of ethanol from barley β-glucan as the sole carbon source by MT8-1/pBG211/pEG23u31H6. Symbols: ▴, ethanol; •, total sugar; ▪, glucose in culture broth. The data points represent the averages of two independent experiments.